Figure 6

PIKfyve is required for starvation-induced mTOR shutdown. (A) HeLa cells were pretreated with DMSO vehicle control or 200 nM Apm for 3 h, followed by starvation in HBSS in the absence or in the presence of 200 nM Apm (HBSS + Apm) for the indicated time. Lysates were probed with the indicated antibodies. (B) Densitometric analysis of phosphorylation status of 4E-BP1 and S6K in (A). Data are represented as mean ± SD of 3 independent experiments (ns, non-significant; ** p < 0.01, *** p < 0.001, **** p < 0.0001; two-way ANOVA). (C) HeLa cells were treated as in (A), fixed at the selected time point 0 (Fed) and 120 min (HBSS 2 h). Cells were stained with the indicated antibodies and visualized by confocal microscopy. Scale bar, 20 µm. (D) Quantification of TSC2/LAMP1 colocalization from (C) (**** p < 0.0001, one-way ANOVA). (E) A working model for PIKfyve-dependent regulation of mTOR activity. The red arrow pointing upward indicates hyperactivation of mTOR activity. The prohibition sign indicates mTOR inhibition.